CN104391019A - Aptamer electrochemical biosensor, as well as preparation method and application thereof - Google Patents
Aptamer electrochemical biosensor, as well as preparation method and application thereof Download PDFInfo
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- CN104391019A CN104391019A CN201410591942.1A CN201410591942A CN104391019A CN 104391019 A CN104391019 A CN 104391019A CN 201410591942 A CN201410591942 A CN 201410591942A CN 104391019 A CN104391019 A CN 104391019A
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Abstract
The invention relates to an aptamer electrochemical biosensor, as well as a preparation method and application therof. The preparation method comprises the following steps: (1) respectively dissolving DNA sequences into a PBS buffer solution; (2) firstly polishing and washing a gold electrode; (3) dispensing the buffer solution containing a probe S1 and a probe S2 onto the gold electrode for culturing; (4) dispensing the PBS buffer solution onto an S2-S1/GE electrode for culturing; (5) placing S2-S2/GE into the PBS buffer solution to be cultured; washing the electrode to obtain a Aptasensor; (6) culturing the acquired Aptasensor in the buffer solution, and thus obtaining a decorative electrode HP2-HP1-Aptasensor of a super-sandwiched structure by virtue of DNA hybrid chain reaction; and (7) culturing the HP2-HP1-Aptasensor in the buffer solution to obtain a silver nano-cluster decorated electrode AgNCs/HP2-HP1-Aptasensor. According to the preparation method of the biosensor, the used nano material is simple to synthesize, the energy consumption is low, the cost is low, the biological compatibility is good, the mimic enzyme catalytic activity of the nano material is studied and used as an electrochemical detection signal; moreover, a signal detected by the prepared biosensor is amplified by utilizing the DNA hybrid chain reaction.
Description
Technical field
The present invention relates to biosensor technology field, be specifically related to the silver nanoclusters with analogue enztme activity to be assembled on electrochemica biological sensor with DNA hybridization chain reaction and to apply this sensor amplification detection targeted fit body lysokinase.
Background technology
In recent years, be an extremely important aspect in life science to the research of DNA, the research of DNA biosensor becomes focus.Having research much related to this to be devoted to is the measured signal of metal nano material (as: gold nanoclusters (AuNCs), silver nanoclusters (AgNCs)) as DNA biosensor of template with DNA, but wherein most research is the optical property using metal nanometre cluster, seldom study the analogue enztme probing into utilization metal nanometre cluster active.In the strategy amplifying DNA biosensor detection signal, DNA hybridization chain reaction, has the inherent advantages such as reaction conditions gentleness, background signal are little, label-free process, can amplify strategy as good signal and be widely used because of it.
Summary of the invention
The object of the present invention is to provide a kind of aptamers electrochemica biological sensor, its preparation method and purposes, especially a kind of preparation of aptamers electrochemica biological sensor of the mimetic enzyme catalysis effect based on DNA hybridization chain reaction and silver nanoclusters and the application of amplification detection aptamers thereof, utilize the specific binding effect of targeted fit body and specific DNA, use DNA hybridization chain reaction at the surface-assembled electrochemica biological sensor of gold electrode, be applied to the detection of targeted fit body molecule.Concrete technical scheme is as follows:
A preparation method for aptamers electrochemica biological sensor, comprises the steps:
(1) DNA sequence dna is dissolved in respectively in PBS buffer solution;
(2) gold electrode (GE) is first carried out polishing and cleaning;
(3) buffer solution containing probe S1 and S2 is dripped be coated onto on gold electrode, cultivate and obtain S2-S1/GE;
(4) the PBS buffer solution containing certain density aptamers is dripped be coated on S2-S1/GE electrode, cultivate; Aptasensor is obtained after cleaning electrode;
(5) Aptasensor of acquisition is cultivated in containing the buffer solution of HP1 and HP2, obtained the modified electrode HP2-HP1-Aptasensor of super sandwich structure by DNA hybridization chain reaction.
(6) HP2-HP1-Aptasensor is cultivated containing in silver ion and sodium borohydride buffer solution, obtain the Electrode Ag NCs/HP2-HP1-Aptasensor that silver nanoclusters is modified.
Further, in step (1), DNA sequence dna comprises: sulfhydrylation DNA:S1, with aptamers specific binding DNA:S2, hair clip DNA 1:HP1, hair clip DNA 2:HP2; Described DNA is dissolved in the PBS buffer solution of pH 7.4 respectively, and saves backup down at low temperatures.
Further, in step (2), comprise the steps:
(2-1) gold electrode is carried out polishing with the aluminium powder of 0.3 μm;
(2-2) gold electrode is carried out polishing with the aluminium powder of 0.5 μm;
(2-3) put into HNO3:H2O (v/v)=1:1 solution, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is 3 ~ 5min;
(2-4) put into ethanolic solution, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is 3 ~ 5min;
(2-5) put into ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is 3 ~ 5min.
Further, in step (3), the buffer solution containing probe S1 and S2 is dripped and is coated onto on the clean gold electrode of surface treatment, cultivate 6-10h, the gold electrode (S2-S1/GE) modified by Au-S covalent bond effect self assembly S1 and S2.
Further, in step (4), the PBS buffer solution containing certain density aptamers is dripped and is coated on S2-S1/GE electrode, after cultivating about 30 ~ 40min, obtain Aptasensor; Modified electrode nitrogen drying after thorough cleaning, saves backup.
Further, in step (5), the Aptasensor of acquisition is cultivated about 10h in containing the buffer solution of HP1 and HP2, obtained the modified electrode HP2-HP1-Aptasensor of super sandwich structure by DNA hybridization chain reaction.
Further, in step (6), in the sequence that the 3 ' end of HP2 is one section of rich cytimidine, HP2-HP1-Aptasensor is cultivated in the buffer solution containing silver ion and sodium borohydride, obtains the electrode (AgNCs/HP2-HP1-Aptasensor) that silver nanoclusters is modified.
A kind of aptamers electrochemica biological sensor, adopts the preparation method as preceding claim aptamers electrochemica biological sensor to obtain.
The purposes of above-mentioned aptamers electrochemica biological sensor, because the concentration of aptamers is different, the amount of the S1DNA chain on the Aptasensor formed will be different, and then will be different by the amount being obtained the AgNCs on super sandwich structure modified electrode by DNA hybridization chain reaction, along with the increase of aptamers concentration, the amount being assembled into the AgNCs on sensor also can increase thereupon, and therefore this sensor quantitatively can detect variable concentrations targeted fit body.
Compared with currently available technology, the preparation method of biology sensor of the present invention, the nano material synthesis used is simple, consume energy low, cost is low, good biocompatibility, probes into and uses the active signal as Electrochemical Detection of the mimetic enzyme catalysis of nano material, and using the signal that the biology sensor prepared by DNA hybridization chain reaction amplification detects.By the specific binding effect between aptamers and specific DNA, prepare a kind of electrochemica biological sensor of unmarked, sensitive, " turn-on " detection aptamers.It is satisfactory that result shows the testing result of this biology sensor to targeted fit body molecule, and the range of linearity of detection is wider, about from 10
-10m is to 10
-5have sensitiveer detection within the scope of M, and have highly sensitive, detectability is low, selectivity good, the feature of good stability.In addition, in view of this electrochemica biological sensor is that the gold electrode (S2-S1/GE) utilizing the specific binding effect between aptamers and specific DNA to be modified by S1 and S2 is assembled into Aptasensor, we also can utilize the base mispairing associated methods of cytimidine-silver ion-cytimidine etc., obtain Aptasensor, therefore this biology sensor is expected to become the realization of a kind of detection platform to the pervasive detection of aptamers (such as atriphos or fibrin ferment etc.) or the Sensitive Detection of small molecule analysis thing (such as silver ion or mercury ion etc.).
Accompanying drawing explanation
Fig. 1 is the schematic diagram prepared based on the aptamers electrochemica biological sensor of the mimetic enzyme catalysis effect of DNA hybridization chain reaction and silver nanoclusters;
Fig. 2 is the transmission electron microscope picture of the AgNCs nano material of preparation;
Fig. 3 (A) is the cyclic voltammogram of silver nanoclusters in the electrochemica biological sensor of assembling;
In figure:
A is the cyclic voltammogram of naked gold electrode.
B is the Aptasensor cyclic voltammogram that AgNCs and S3, HP1 modify;
C is the Aptasensor cyclic voltammogram that AgNCs and HP2, HP1 modify;
Fig. 3 (B) is the impedance diagram of each step electrode modification process electrode;
In figure:
A is the impedance diagram of naked gold electrode.
B is the impedance diagram of the gold electrode that S2 and S1 modifies.
C is the impedance diagram of the Aptasensor obtained after S2-S1/GE cultivates in the damping fluid containing aptamers.
D is the Aptasensor impedance diagram that HP2, HP1 modify;
E is the Aptasensor impedance diagram that AgNCs and HP2, HP1 modify;
Fig. 3 (C) is the cyclic voltammogram of different modifying electrode pair hydrogen peroxide catalysis;
In figure:
A is the cyclic voltammogram of naked gold electrode;
B is the cyclic voltammogram of the gold electrode that S2 and S1 modifies;
C is the cyclic voltammogram of the Aptasensor obtained after S2-S1/GE cultivates in the damping fluid containing aptamers;
D is the cyclic voltammogram of the Aptasensor that AgNCs and HP2, HP1 modify;
E is the cyclic voltammogram of the Aptasensor that AgNCs and S3, HP1 modify;
F is the cyclic voltammogram of the Aptasensor that HP2, HP1 modify;
Fig. 4 (A) is to the cyclic voltammogram of variable concentrations hydrogen peroxide catalysis based on the aptamers electrochemica biological sensor of the mimetic enzyme catalysis effect of DNA hybridization chain reaction and silver nanoclusters;
Fig. 4 (B) for this reason sensor to the typical curve of variable concentrations hydrogen peroxide catalysis.
Fig. 5 (A) is to the cyclic voltammogram of variable concentrations targeted fit body Molecular Detection based on the aptamers electrochemica biological sensor of the mimetic enzyme catalysis effect of DNA hybridization chain reaction and silver nanoclusters;
Fig. 5 (B) is to the chronoamperogram of variable concentrations targeted fit body Molecular Detection based on the aptamers electrochemica biological sensor of the mimetic enzyme catalysis effect of DNA hybridization chain reaction and silver nanoclusters;
Fig. 5 (C) be based on the mimetic enzyme catalysis effect of DNA hybridization chain reaction and silver nanoclusters aptamers electrochemica biological sensor to variable concentrations targeted fit body Molecular Detection typical curve.
Embodiment
Describe the present invention with reference to the accompanying drawings below, it is a kind of preferred embodiment in numerous embodiments of the present invention.
Comprise the steps: based on the preparation of aptamers electrochemica biological sensor of the mimetic enzyme catalysis effect of DNA hybridization chain reaction and silver nanoclusters and the application of amplification detection aptamers thereof
A, the DNA sequence dna (sulfhydrylation DNA:S1, with aptamers specific binding DNA:S2, hair clip DNA 1:HP1, hair clip DNA 2:HP2) bought to be dissolved in PBS (pH 7.4) buffer solution respectively, and to save backup down at low temperatures.
B, gold electrode is first carried out polishing with the aluminium powder of 0.3 and 0.5 μm successively, then put into HNO successively
3: H
2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3 ~ 5min.
C, the buffer solution containing probe S1 and S2 dripped be coated onto on the clean gold electrode of surface treatment, cultivate 6-10h, the gold electrode (S2-S1/GE) modified by Au-S covalent bond effect self assembly S1 and S2.Then being dripped by the PBS buffer solution containing mercaptoethanol is coated on S2-S1/GE electrode, cultivates about 30 ~ 40min.Modified electrode nitrogen drying after thorough cleaning, saves backup.
D, be placed on S2-S1/GE containing certain density aptamers PBS buffer solution in cultivate about 30 ~ 40min, obtain Aptasensor after cleaning electrode.Then the Aptasensor of acquisition is cultivated about 10h in containing the buffer solution of HP1 and HP2, obtained the modified electrode (HP2-HP1-Aptasensor) of super sandwich structure by DNA hybridization chain reaction.Due to the special design of HP2, in the sequence that the 3 ' end of HP2 is one section of rich cytimidine, HP2-HP1-Aptasensor is cultivated in the buffer solution containing negative ion and sodium borohydride, the electrode (AgNCs/HP2-HP1-Aptasensor) that silver nanoclusters is modified can be obtained.Aptamers electrochemica biological sensor based on the mimetic enzyme catalysis effect of DNA hybridization chain reaction and silver nanoclusters is successfully prepared.
E, due to the concentration of aptamers different, the amount of the S1DNA chain on the Aptasensor formed will be different, and then will be different by the amount being obtained the AgNCs on super sandwich structure modified electrode by DNA hybridization chain reaction, along with the increase of aptamers concentration, the amount being assembled into the AgNCs on sensor also can increase thereupon, and therefore this sensor quantitatively can detect variable concentrations targeted fit body.
Above by reference to the accompanying drawings to invention has been exemplary description; obvious specific implementation of the present invention is not subject to the restrictions described above; as long as have employed the various improvement that method of the present invention is conceived and technical scheme is carried out; or directly apply to other occasion, all within protection scope of the present invention without improving.
Claims (9)
1. a preparation method for aptamers electrochemica biological sensor, is characterized in that, comprises the steps:
(1) DNA sequence dna is dissolved in respectively in PBS buffer solution;
(2) gold electrode (GE) is first carried out polishing and cleaning;
(3) buffer solution containing probe S1 and S2 is dripped be coated onto on gold electrode, cultivate and obtain S2-S1/GE;
(4) the PBS buffer solution containing certain density aptamers is dripped be coated on S2-S1/GE electrode, cultivate; Aptasensor is obtained after cleaning electrode;
(5) Aptasensor of acquisition is cultivated in containing the buffer solution of HP1 and HP2, obtained the modified electrode HP2-HP1-Aptasensor of super sandwich structure by DNA hybridization chain reaction;
(6) HP2-HP1-Aptasensor is cultivated containing in silver ion and sodium borohydride buffer solution, obtain the Electrode Ag NCs/HP2-HP1-Aptasensor that silver nanoclusters is modified.
2. the preparation method of aptamers electrochemica biological sensor as claimed in claim 1, it is characterized in that, in step (1), DNA sequence dna comprises: sulfhydrylation DNA:S1, with aptamers specific binding DNA:S2, hair clip DNA 1:HP1, hair clip DNA 2:HP2; Described DNA is dissolved in the PBS buffer solution of pH 7.4 respectively, and saves backup down at low temperatures.
3. the preparation method of aptamers electrochemica biological sensor as claimed in claim 1 or 2, is characterized in that, in step (2), comprise the steps:
(2-1) gold electrode is carried out polishing with the aluminium powder of 0.3 μm;
(2-2) gold electrode is carried out polishing with the aluminium powder of 0.5 μm;
(2-3) HNO is put into
3: H
2o (v/v)=1:1 solution, carries out Ultrasonic Cleaning, and the time of ultrasonic cleaning is 3 ~ 5min;
(2-4) put into ethanolic solution, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is 3 ~ 5min;
(2-5) put into ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is 3 ~ 5min.
4. the preparation method of the aptamers electrochemica biological sensor according to any one of claim 1-3, it is characterized in that, in step (3), buffer solution containing probe S1 and S2 is dripped and is coated onto on the clean gold electrode of surface treatment, cultivate 6-10h, the gold electrode S2-S1/GE modified by Au-S covalent bond effect self assembly S1 and S2.
5. the preparation method of the aptamers electrochemica biological sensor according to any one of claim 1-4, it is characterized in that, in step (4), being dripped by PBS buffer solution containing certain density aptamers is coated on S2-S1/GE electrode, obtains Aptasensor after cultivating about 30 ~ 40min; Modified electrode nitrogen drying after thorough cleaning, saves backup.
6. the preparation method of the aptamers electrochemica biological sensor according to any one of claim 1-5, it is characterized in that, the Aptasensor of acquisition is cultivated about 10h in containing the buffer solution of HP1 and HP2, is obtained the modified electrode HP2-HP1-Aptasensor of super sandwich structure by DNA hybridization chain reaction.
7. the preparation method of the aptamers electrochemica biological sensor according to any one of claim 1-6, it is characterized in that, in step (6), in the sequence that the 3 ' end of HP2 is one section of rich cytimidine, HP2-HP1-Aptasensor is cultivated in the buffer solution containing silver ion and sodium borohydride, obtains the Electrode Ag NCs/HP2-HP1-Aptasensor that silver nanoclusters is modified.
8. an aptamers electrochemica biological sensor, is characterized in that, adopts the preparation method of aptamers electrochemica biological sensor as described in claim 1-7 to obtain.
9. the purposes of aptamers electrochemica biological sensor as claimed in claim 8, it is characterized in that, because the concentration of aptamers is different, the amount of the S1DNA chain on the Aptasensor formed will be different, and then will be different by the amount being obtained the AgNCs on super sandwich structure modified electrode by DNA hybridization chain reaction, along with the increase of aptamers concentration, the amount being assembled into the AgNCs on sensor also can increase thereupon, and therefore this sensor quantitatively can detect variable concentrations targeted fit body.
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| CN106770553B (en) * | 2016-12-20 | 2018-11-30 | 中国科学院苏州生物医学工程技术研究所 | The detection method of platelet derived growth factor |
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| CN109991202A (en) * | 2019-04-16 | 2019-07-09 | 南京医科大学 | A method of it is detected based on aptamer fluorescent optical sensor for multiple target objects |
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